Process for producing matsutake mushroom mycelium

ABSTRACT

Disclosed is a process for producing a mycelium of a  Matsutake  fungus, comprising the step of cultivating a mycelium on a small scale under agitation without aeration or with aeration at a low rate of less than 0.05 vvm in a liquid medium. According to the process, a large number of  mycelia  can be produced without a loss of physiological activities.

TECHNICAL FIELD

The present invention relates to a process for producing mycelia of aMatsutake fungus (Matsutake mycelia). In particular, the presentinvention relates to a process for producing Matsutake mycelia useful asa seed culture for a large-scale production of Matsutake mycelia, and aprocess for producing a large number of Matsutake mycelia using the seedculture.

BACKGROUND ART

International Publication WO02/30440 (patent reference 1) discloses thatdry powder of Tricholoma matsutake mycelia is useful in promoting arecovery from stress (page 27).

Japanese Examined Patent Publication (Kokoku) No. 61-53032 (patentreference 2) discloses a process for producing Tricholoma matsutakemycelia comprising the steps of cultivating a slant culture ofTricholoma matsutake mycelia in a liquid medium containing starch undershaking for 30 days, inoculating a liquid medium (20 L) containingstarch, and carrying out a further cultivation under aeration andagitation for 30 days (Example 1).

Further, Japanese Unexamined Patent Publication (Kokai) No. 11-318433(patent reference 3) discloses a process for producing Tricholomamatsutake mycelia comprising the steps of cultivating mycelia isolatedfrom a commercially available fruit body of Tricholoma matsutake on aplate for 4 days, carrying out a cultivation for acclimation in a liquidmedium containing vegetable extracts for 4 days, and carrying out afurther cultivation in a liquid medium (2 L) containing vegetableextracts using a tank for submerged cultivation under aeration andagitation for 6 days (Example 1).

However, a new process for a large-scale production in which a largenumber of Tricholoma matsutake mycelia can be produced without a loss ofphysiological activities is desired. (patent reference 1) InternationalPublication WO02/30440 (patent reference 2) Japanese Examined PatentPublication (Kokoku) No. 61-53032 (patent reference 3) JapaneseUnexamined Patent Publication (Kokai) No. 11-318433

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a process for producingmycelia of a Matsutake fungus (Matsutake mycelia) useful as a seedculture for a large-scale production of Matsutake mycelia, and a processfor producing a large number of Matsutake mycelia, using the seedculture, without a loss of physiological activities.

The present inventors have conducted intensive studies and, as a result,found that a seed culture for a large-scale production of Tricholomamatsutake mycelia can be obtained by cultivating Tricholoma matsutakemycelia on a small scale under agitation without aeration or withaeration at a low rate in a liquid medium. Preferably, the Tricholomamatsutake mycelia used in the agitation cultivation is produced bycultivating initial mycelia (previously cultivated or maintained in asolid or liquid medium) in a liquid medium under stationary conditionsfor an appropriate period, and further cultivating the obtained myceliaunder shaking. Further, the present inventors found that a large numberof Tricholoma matsutake mycelia can be efficiently produced bycultivating the seed culture under submerged conditions (for example,submerged cultivation under agitation).

The present invention relates to: [1] a process for producing a myceliumof a Matsutake fungus, comprising the step of cultivating a mycelium ona small scale under agitation without aeration or with aeration at a lowrate of less than 0.05 vvm in a liquid medium (hereinafter referred toas agitating cultivation without aeration); [2] the process of [1],further comprising, as a precultivation step before the agitatingcultivation step, (1) cultivating a mycelium in a liquid medium understationary conditions, (2) cultivating a mycelium under shaking, or (3)cultivating a mycelium in a liquid medium under stationary conditions,and further cultivating the obtained mycelium under shaking; [3] theprocess of [1] or [2], wherein an agitation power per unit volume of aculture medium in the agitating cultivation step is 0.01 to 2 kW/m³; [4]a process for producing a mycelium of a Matsutake fungus (particularly,a seed culture for producing a mycelium of a Matsutake fungus),comprising the steps of: cultivating a mycelium in a liquid medium understationary conditions, and cultivating the obtained mycelium undershaking; [5] a process for producing a mycelium of a Matsutake fungus,comprising the step of cultivating the mycelium obtained by the processof any one of [1] to [4] as a seed culture under submerged conditions;[6] the process of any one of [2] to [5], wherein a period of thestationary cultivation step is 30 to 400 days; [7] the process of anyone of [2] to [6], wherein a period of the shaking cultivation step is 5to 50 days; [8] the process of any one of [1] to [7], wherein apropagation rate in inoculation is 2 to 50 times; [9] the process of anyone of [1] to [8], wherein an osmotic pressure of a culture medium is0.01 to 0.8 MPa; and [10] the process of any one of claim [1] to [9],wherein a concentration of a fibrous mycelium contained in an initialmycelium is 0.05 g/L or more.

According to a preferred embodiment of the submerged cultivation step inthe process of [5], the mycelium of a Matsutake fungus produced by theprocess of any one of [1] to [3] is used as the seed culture to carryout submerged cultivation on a large scale.

Further, the present invention relates to a process for producing amycelium of a Matsutake fungus, comprising the steps of: cultivating amycelium cultivated or maintained in a solid or liquid medium, in aliquid medium under stationary conditions, and cultivating the obtainedmycelium under shaking.

Further, the present invention relates to a process for producing amycelium of a Matsutake fungus, comprising the steps of: cultivating amycelium cultivated or maintained in a solid or liquid medium, in aliquid medium under stationary conditions, cultivating the obtainedmycelium under shaking, and carrying out an agitating cultivationwithout aeration in a liquid medium using a small-sized fermentor ofless than 100 L.

Further, the present invention relates to a process for producing amycelium of a Matsutake fungus, comprising the steps of: cultivating amycelium cultivated or maintained in a solid or liquid medium, in aliquid medium under stationary conditions, cultivating the obtainedmycelium under shaking, carrying out agitating cultivation withoutaeration in a liquid medium using a small-sized fermentor of less than100 L to produce a seed culture, and cultivating the obtained seedculture under submerged conditions using a middle-sized or large-sizedfermentor of 100 L or more.

The terms “small-scale cultivation or production” and “large-scalecultivation or production” as used herein are well-known meanings usedin well-known stepwise cultivation methods for producing a large numberof microorganisms, in which the initial cultivation is started with asmall-scale culture medium, and stepwisely transferred to cultivationswith a large-scale culture medium. Therefore, each range of“small-scale” or “large-scale” is not absolutely defined by a specificvolume of culture medium, but is a relative concept which can beappropriately determined in accordance with, for example, scale-upprocedures (particularly a volume of a fermentor).

Similarly, the terms “small-sized fermentor” and “large-sized fermentor”as used herein are not absolutely defined by specific volumes thereof,but are relative concepts which can be appropriately determined inaccordance with, for example, scale-up procedures (particularly a scaleof cultivation).

In this connection, preferable ranges of the terms will be illustratedin detail hereinafter.

The following expressions will be used hereinafter to facilitate anunderstanding of the present invention:

The initial strain of a Matsutake fungus is referred to as Matsutake I.

Mycelia of a Matsutake fungus obtained by cultivating or maintaining theMatsutake I in a solid or liquid medium are referred to as Matsutake II.

Mycelia of a Matsutake fungus obtained by cultivating the Matsutake IIin a liquid medium under stationary conditions are referred to asMatsutake III.

Mycelia of a Matsutake fungus obtained by cultivating the Matsutake IIIunder shaking are referred to as Matsutake IV.

Mycelia of a Matsutake fungus obtained by cultivating the Matsutake IVon a small scale under agitation without aeration or with aeration at alow rate in a liquid medium using a small-sized fermentor (for example,a small-sized fermentor having a volume of less than 100 L) are referredto as Matsutake V.

Mycelia of a Matsutake fungus obtained by cultivating the Matsutake Vunder submerged conditions on a large scale or using a large-sizedfermentor (for example, a large-sized fermentor having a volume of 100 Lor more) are referred to as Matsutake VI.

Mycelia of a Matsutake fungus obtained by cultivating the Matsutake VIunder submerged conditions on a large scale or using a large-sizedfermentor (for example, a large-sized fermentor having a volume of 100 Lor more) are referred to as Matsutake VII.

Mycelia of a Matsutake fungus obtained by cultivating the Matsutake VIIunder submerged conditions on a large scale or using a large-sizedfermentor (for example, a large-sized fermentor having a volume of 100 Lor more) are referred to as Matsutake VIII.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the relationship between an agitation powerand growth when the cultivation is carried out at 23±1° C. using a 200-Lfermentor and a medium culture containing starch, glucose, potassiumdihydrogen phosphate, and the like. The symbols “solid circle”, “solidsquare”, and “solid triangle” indicate the results at the agitationpowers of 0.12 kw/m³, 1.1 kw/m³, and 2.6 kw/m³, respectively.

FIG. 2 is a graph showing the relationship between an osmotic pressureand growth when the cultivation is carried out at 23±1° C. using a 200-Lfermentor (agitation power=0.12 kw/m³) and a medium culture containingstarch, glucose, potassium dihydrogen phosphate, and the like. Thesymbols “solid circle”, “solid square”, and “solid triangle” indicatethe results at the osmotic pressures of 0.98 MPa, 0.5 MPa, and 0.05 MPa,respectively.

FIG. 3 is a graph showing the relationship between a concentration ofinitial mycelia and growth when the cultivation is carried out at 23±1°C. using a 500-mL flask with agitation (agitation power=0.14 kw/m³) anda medium culture containing starch, glucose, potassium dihydrogenphosphate, and the like. The symbols “solid circle”, “solid square”,“solid triangle”, and “X” indicate the results at the concentrations ofinitial mycelia of 0.06 g/L, 0.2 g/L, 0.6 g/L, and 1 g/L, respectively.

FIG. 4 shows a form of fibrous mycelia.

FIG. 5 shows a form of pelleted mycelia.

FIG. 6 is a graph showing the relationship between a form of a cultureseed and growth when the cultivation is carried out at 23±1° C. using a200-L fermentor (agitation power=0.12 kw/m³; volume of culturemedium=140 L) and a medium culture containing starch, glucose, potassiumdihydrogen phosphate, and the like. The symbols “solid circle” and“solid square” indicate the results when the seed cultures were fibrousmycelia and pelleted mycelia, respectively.

FIG. 7 shows the outline of a cultivation system for a seed culture.

FIG. 8 shows a cultivation system capable of carrying out the process ofthe present invention.

FIG. 9 is a graph showing the relationship between a cultivation periodand a mycelia content when the cultivation is carried out at 23±1° C.using a 65-m³ fermentor (agitation power=0.01 to 0.12 kw/m³; volume ofculture medium=40 m³) and a medium culture containing starch, glucose,potassium dihydrogen phosphate, and the like.

BEST MODE FOR CARRYING OUT THE INVENTION

In the process of the present invention, mycelia of a Matsutake funguscan be produced by carrying out at least the agitating cultivation stepwithout aeration. The term “agitating cultivation without aeration” asused herein means that a cultivation without aeration or with aerationthereto at a rate of 0.05 vvm or less (i.e., without substantialaeration) in a liquid phase (i.e., a liquid medium) under agitating. Inthis connection, the term “agitating cultivation without aeration”includes a case in which aeration in a gas phase is carried out tomaintain an internal pressure of a fermentor. Mycelia of a Matsutakefungus produced by the agitating cultivation step without aeration maybe a final product, or may be used as a seed culture for a large-scaleproduction. It is preferable to use the mycelia as the seed culture fora large-scale production.

As mycelia of a Matsutake fungus (Matsutake mycelia) which may be usedin the agitating cultivation step without aeration in the process of thepresent invention, there may be mentioned, for example, Matsutakemycelia obtained by carrying out an appropriate precultivation for thepurpose of, for example, growth or acclimation. As the precultivation,there may be mentioned, for example, stationary liquid cultivation orshaking cultivation, or a combination thereof (particularly, successivecultivation of stationary liquid cultivation and shaking cultivation).

Hereinafter, the process of the present invention will be explained inthe sequential order of stationary liquid cultivation, shakingcultivation, agitating cultivation without aeration, and submergedcultivation.

The term “Matsutake fungus” as used herein means a fungus belonging togenus Tricholoma, including Tricholoma matsutake and relatives thereof,such as Tricholoma fulvocastaneum Hongo sp.nov., Tricholomabakamatsutake Hongo sp.nov., or Tricholoma robustum Ricken.

As a Matsutake fungus (for example, Matsutake I as the initial strain ofa Matsutake fungus) used in the process of the present invention,mycelia isolated from a naturally-occurring or commercially availablefruit body may be used.

Further, a strain commercially available or deposited in, for example,the International Patent Organism Depositary National Institute ofAdvanced Industrial Science and Technology may be used in the process ofthe present invention. As the strain, there may be mentioned, forexample, strains ATCC34979, ATCC34981, and ATCC34988 deposited inAmerican Type Culture Collection (ATCC), strains IFO6915, IFO6925,IFO6930, IFO6935, IFO30604, IFO30605, and IFO30606 deposited inInstitute for Fermentation, Osaka (IFO), strain MAFF460038 deposited inNational Institute of Agrobiological Sciences, and Tricholoma matsutakestrain FERM BP-7304 deposited in the International Patent OrganismDepositary National Institute of Advanced Industrial Science andTechnology.

A medium used in cultivating or maintaining Matsutake II is notparticularly limited, so long as it contains the substrates for nutrientsource commonly used in cultivating a Matsutake fungus. As the medium,there may be mentioned, for example, Ohta medium [Ohta, A., (1990)Trans. Mycol. Soc. Japan 31: 323-334], MMN medium [Marx, D. H. (1969)Phytopathology 59: 153-163], or Hamada medium [Hamada, (1964) Matsutake,97-100].

As an agent for solidifying a solid medium, there may be mentioned, forexample, carageenan, mannan, pectin, agar, curdlan, starch, or alginate.Agar is preferable.

As the substrates for nutrient source, for example, carbon sources,nitrogen sources, or inorganic element sources may be used.

As the carbon sources, there may be mentioned, for example, starch (suchas rice starch, wheat starch, potato starch, or sweet potato starch),polysaccharides (such as dextran or amylopectin), oligosaccharides (suchas maltose or sucrose), monosaccharides (such as fructose or glucose),or malt extract. There is a period in which monosaccharides such asglucose is preferable and a period in which starch is preferable, inaccordance with a growth rate of a Matsutake fungus. Therefore, one ormore appropriate carbon sources may be selected in accordance with suchperiods, and, if necessary, a combination thereof can be used.

As the nitrogen sources, there may be mentioned, for example, yeastextract, dried yeast, corn steep liquor, soybean powder, or soybeanpeptone, which are derived from natural substances. Further, ammoniumnitrate, ammonium sulfate, or urea may be used. The above nitrogensources may be used alone or in a combination thereof. Substancesderived from natural substances (particularly yeast extract) aregenerally preferable in the light of a growth rate.

The inorganic element sources are used to supply phosphates and traceelements. There may be mentioned, for example, phosphates or inorganicsalts such as sulfates, hydrochlorides, nitrates, or phosphates of metalions (for example, sodium, potassium, magnesium, calcium, zinc,manganese, copper, or iron). The required amount is dissolved in aculture medium.

Further, vitamins such as vitamin B1 or amino acids may be added to aculture medium.

Furthermore, for example, plant extracts, organic acids, or nucleic acidrelated substances may be added in accordance with the properties of aMatsutake fungus used. As the plant extracts, there may be mentioned,for example, extracts of fruit vegetables, root vegetables, or leafvegetables. As the organic acids, there may be mentioned, for example,citric acid, tartaric acid, malic acid, fumaric acid, or lactic acid. Asthe nucleic acid related substances, there may be mentioned, forexample, commercially available nucleic acid, nucleic acid extract,yeast, or yeast extract.

When a solid medium is prepared, the content of the carbon sources ispreferably 10 to 100 g/L, more preferably 10 to 50 g/L, most preferably20 to 30 g/L.

The content of the nitrogen sources (as the amount of the nitrogenelement converted) is preferably 0.005 to 0.1 mol/L, more preferably0.007 to 0.07 mol/L, most preferably 0.01 to 0.05 mol/L.

The content of phosphates (as the amount of the phosphorus elementconverted) is preferably 0.001 to 0.05 mol/L, more preferably 0.005 to0.03 mol/L, most preferably 0.01 to 0.02 mol/L. The other inorganicsalts, vitamins, plant extracts, organic acids, and/or nucleic acidrelated substances may be appropriately added in accordance with theproperties of a Matsutake fungus. Further, the pH of a prepared solutioncontaining substances for nutrient source is preferably pH 4 to 7, morepreferably pH 4.5 to 6.0, most preferably pH 5.0 to 5.5.

(Stationary Liquid Cultivation)

The process for producing Matsutake III by cultivating Matsutake II(Matsutake fungus cultivated or maintained in a solid or liquid medium)in a liquid medium under stationary conditions will be explained.

In the process, a culture vessel (for example, a 30-mL to 10-L conicalflask, preferably a 100-mL to 2-L conical flask) capable of carrying outa stationary liquid cultivation is generally used.

The stationary liquid cultivation is started by inoculating a liquidmedium with Matsutake II.

As to a volume of the liquid medium, a ratio (Vt/Vo; hereinafterreferred to as “propagation rate in inoculation”) of a total volume (Vt)of a broth containing Matsutake II for inoculation and the liquid mediumwith respect to a volume of the broth (Vo) is preferably 2 to 50 times,more preferably 3 to 30 times.

In the inoculation of the liquid medium with the broth containingMatsutake II, a ratio (Wo/Vt; hereinafter referred to as “concentrationof initial mycelia”) of a dried weight (Wo) of mycelia of Matsutake IIcontained in the broth containing Matsutake II for inoculation withrespect to a volume (Vt) of the mixture of the broth and the liquidmedium is preferably 0.05 to 3 g/L, more preferably 0.1 to 2 g/L.

In the stationary liquid cultivation, the cultivation is carried out atpreferably 15 to 30° C., more preferably 20 to 25° C. for preferably 30to 400 days, more preferably 120 to 240 days. When the period for thestationary liquid cultivation is less than 30 days or more than 400days, it becomes difficult to obtain Matsutake III having a viabilitysuitable for a large-scale cultivation.

The liquid medium used in the stationary liquid cultivation containssubstrates for nutrient source, and thus an osmotic pressure of theliquid medium is preferably 0.01 to 0.8 MPa, more preferably 0.02 to 0.7MPa, most preferably 0.03 to 0.5 MPa.

As the substrates for nutrient source used in the stationary liquidcultivation, the same carbon sources, nitrogen sources, inorganicelement sources, vitamins (such as vitamin B1), or amino acids as theabove-mentioned solid medium for cultivating Matsutake I can be used.

The content of the carbon sources is preferably 10 to 100 g/L, morepreferably 20 to 60 g/L, most preferably 25 to 45 g/L. Monosaccharidessuch as glucose are generally used.

The content of the nitrogen sources (as the amount of the nitrogenelement converted) is preferably 0.005 to 0.1 mol/L, more preferably0.007 to 0.07 mol/L, most preferably 0.01 to 0.05 mol/L.

When phosphates are used, the content thereof (as the amount of thephosphorus element converted) is preferably 0.001 to 0.05 mol/L, morepreferably 0.005 to 0.03 mol/L, most preferably 0.01 to 0.02 mol/L.

The other inorganic salts, vitamins, plant extracts, organic acids, andnucleic acid related substances may be appropriately added in accordancewith the properties of a Matsutake fungus.

Further, the pH of a prepared solution containing substances fornutrient source is preferably pH 4 to 7, more preferably pH 4.5 to 6.5,most preferably pH 5.0 to 6.0.

In the stationary liquid cultivation, the starting culture medium can beinoculated with a portion or the whole of the Matsutake III-containingbroth previously obtained by another stationary liquid cultivation, aswell as with the Matsutake II-containing broth as described above.

(Shaking Cultivation)

The process for producing Matsutake IV by cultivating Matsutake IIIunder shaking will be explained.

In the process, a culture vessel (for example, a 30-mL to 10-L,preferably a 300-mL to 5-L conical flask or shaking flask capable ofcarrying out shaking cultivation is generally used.

The shaking cultivation can be started by inoculating a liquid mediumwith Matsutake III (i.e., Matsutake fungus obtained by the stationaryliquid cultivation), or with Matsutake II (i.e., Matsutake funguscultivated or maintained in a solid or liquid medium). Before theinoculation, it is preferable to homogenize the Matsutake mycelia (i.e.,Matsutake III or Matsutake II) used for inoculation.

As to a volume of the liquid medium, a ratio (hereinafter referred to as“propagation rate in inoculation”) of a total volume of a brothcontaining Matsutake III for inoculation and the liquid medium withrespect to a volume of the broth is preferably 2 to 50 times, morepreferably 3 to 30 times, most preferably 5 to 10 times.

In this connection, the stationary liquid cultivation may be carried outusing plural culture vessels to prepare a sufficient amount of brothrequired for an appropriate propagation rate in inoculation.

In the inoculation of the liquid medium with the broth containingMatsutake III, a ratio (hereinafter referred to as “concentration ofinitial mycelia”) of a dried weight of mycelia of Matsutake IIIcontained in the broth containing Matsutake III for inoculation withrespect to a volume of the mixture of the broth and the liquid medium ispreferably 0.05 to 3 g/L, more preferably 0.1 to 2 g/L.

The shaking cultivation is carried out at preferably 15 to 30° C., morepreferably 20 to 25° C. for preferably 5 to 50 days, more preferably 7to 50 days, most preferably 14 to 28 days to produce Matsutake IV usedfor the agitating cultivation.

In the shaking cultivation, an agitation power per unit volume of aculture medium contained in a conical flask is generally 0.01 to 2kW/m³, preferably 0.05 to 0.4 kW/m³.

The liquid medium used in the shaking cultivation contains substratesfor nutrient source, and thus an osmotic pressure of the liquid mediumis preferably 0.01 to 0.8 MPa, more preferably 0.02 to 0.7 MPa, mostpreferably 0.03 to 0.5 MPa.

As the substrates for nutrient source used in the shaking cultivation,the same carbon sources, nitrogen sources, inorganic element sources,vitamins (such as vitamin B1), or amino acids as the above-mentionedliquid medium for cultivating Matsutake II can be used.

The content of the carbon sources is preferably 10 to 100 g/L, morepreferably 20 to 60 g/L, most preferably 25 to 45 g/L. Monosaccharidessuch as glucose are generally used.

The content of the nitrogen sources (as the amount of the nitrogenelement converted) is preferably 0.005 to 0.1 mol/L, more preferably0.007 to 0.07 mol/L, most preferably 0.01 to 0.05 mol/L.

The content of phosphates (as the amount of the phosphorus elementconverted) is preferably 0.001 to 0.05 mol/L, more preferably 0.005 to0.03 mol/L, most preferably 0.01 to 0.02 mol/L.

The other inorganic salts, vitamins, amino acids, plant extracts,organic acids, and/or nucleic acid related substances may beappropriately added in accordance with the properties of a Matsutakefungus.

Further, the pH of a prepared solution containing substances fornutrient source is preferably pH 4 to 7, more preferably pH 4.5 to 6.5,most preferably pH 5.0 to 6.0.

(Agitating Cultivation Without Aeration)

The process for producing Matsutake V by agitating cultivation withoutaeration will be explained.

The agitating cultivation is started by inoculating a liquid medium withMatsutake IV (i.e., Matsutake fungus obtained by the shakingcultivation) or with Matsutake III (i.e., Matsutake fungus obtained bythe stationary liquid cultivation). Before the inoculation, theMatsutake mycelia (i.e., Matsutake III or Matsutake II) used forinoculation can be homogenized.

Hereinafter, the agitating cultivation step without aeration will beexplained by an embodiment in which the liquid medium is inoculated withMatsutake IV, but an embodiment in which the liquid medium is inoculatedwith Matsutake III can be similarly carried out. Further, mycelia of aMatsutake fungus obtained by the agitating cultivation can be used torepeat the agitating cultivation.

The liquid medium used in the agitating cultivation without aeration maybe prepared as follows:

As the substrates for nutrient source, the same carbon sources, nitrogensources, inorganic element sources, vitamins (such as vitamin B1), oramino acids as the above-mentioned liquid medium for shaking cultivationcan be used.

The content of the carbon sources is preferably 10 to 100 g/L, morepreferably 20 to 60 g/L, most preferably 25 to 45 g/L. Starch may bepreferably used.

When monosaccharides (such as glucose), which affect an osmotic pressureof the liquid medium for cultivating under agitation without aeration,are used together with starch, the content of the monosaccharides ispreferably 0.1 to 60 g/L, more preferably 0.5 to 40 g/L, most preferably0.7 to 20 g/L.

The content of the nitrogen sources (as the amount of the nitrogenelement converted) is preferably 0.005 to 0.1 mol/L, more preferably0.007 to 0.07 mol/L, most preferably 0.01 to 0.05 mol/L.

The content of phosphates (as the amount of the phosphorus elementconverted) is preferably 0.001 to 0.05 mol/L, more preferably 0.005 to0.03 mol/L, most preferably 0.01 to 0.02 mol/L.

The other inorganic salts, vitamins, amino acids, plant extracts,organic acids, and/or nucleic acid related substances may beappropriately added in accordance with the properties of a Matsutakefungus.

Further, the pH of a prepared solution containing substances fornutrient source is preferably pH 4 to 7, more preferably pH 4.5 to 6.5,most preferably pH 5.0 to 6.0.

The liquid medium used in the agitating cultivation without aerationcontains substrates for nutrient source, and thus an osmotic pressure ofthe liquid medium is preferably 0.01 to 0.8 MPa, more preferably 0.02 to0.7 MPa, most preferably 0.03 to 0.5 MPa.

The temperature in the agitating cultivation without aeration ispreferably 15 to 30° C., more preferably 20 to 25° C.

As to a volume of the liquid medium, a ratio (hereinafter referred to as“propagation rate in inoculation”) of a total volume of a brothcontaining Matsutake IV for inoculation and the liquid medium withrespect to a volume of the broth is preferably 2 to 50 times, morepreferably 3 to 30 times, most preferably 5 to 10 times.

In the inoculation of the liquid medium with the broth containingMatsutake IV, a ratio (hereinafter referred to as “concentration ofinitial mycelia”) of a dried weight of mycelia of Matsutake IV containedin the broth containing Matsutake IV for inoculation with respect to avolume of the mixture of the broth and the liquid medium is preferably0.01 to 5 g/L, more preferably 0.05 to 3 g/L, most preferably 0.1 to 2g/L. In this connection, it is preferable that a concentration offibrous mycelia contained in the initial mycelium is 0.005 to 5 g/L,more preferably 0.025 to 3 g/L, most preferably 0.05 to 2 g/L.

When Matsutake V obtained in the agitating cultivation without aerationare used as a seed culture for the following submerged cultivation, aperiod of the agitating cultivation without aeration is preferably 3 to20 days, more preferably 5 to 14 days.

The broth in which a dried weight of mycelia of Matsutake V ispreferably 0.5 to 10 g/L, more preferably 1 to 8 g/L, most preferably 1to 6 g/L after the above cultivation period contains Matsutake V havinga viability suitable for submerged cultivation.

When Matsutake V obtained in the agitating cultivation without aerationare separated as a final product, a period of the agitating cultivationwithout aeration is preferably 5 to 30 days, more preferably 7 to 20days, most preferably 10 to 15 days.

After the above cultivation period, the cultivation can be finished,preferably when a rate of utilizing carbon sources is remarkablydecreased. The cultivation period can be appropriately selected inaccordance with a production process including a production cycle orcost.

In the shaking cultivation and the subsequent cultivations, the obtainedmycelia are broadly classified into fibrous mycelia and pelletedmycelia.

In the process of the present invention, when mycelia produced in eachstep is used as a seed culture for the subsequent step, myceliacontaining 50% or more (more preferably 80% or more) of fibrous myceliais preferable. When a liquid medium is inoculated with fibrous mycelia,a rapid growth is observed, in comparison with pelleted mycelia. In thisconnection, when a sufficient amount of the whole mycelia can beprepared, even if the content of fibrous mycelia contained in the wholemycelia is less than the above percentage, a sufficient amount offibrous mycelia is contained therein, and thus, the percentage offibrous mycelia is not particularly limited. The ratio of fibrousmycelia to pelleted mycelia may be determined, for example, by passingthe mycelia mixture through a mesh filter having a mesh opening ofapproximately 1 mm.

In contrast, when mycelia obtained in a certain step is separated as afinal product, the ratio of fibrous mycelia to pelleted mycelia afterthe final step is not important and is not particularly limited, becauseit is important to collect a sufficient amount of mycelia as the whole.

The agitating cultivation without aeration is carried out on a smallscale, i.e., using a small-scale culture medium. A volume of the mediumused in the agitating cultivation without aeration is not particularlylimited, so long as it does not exceed a range of a small-scalecultivation in an ordinary stepwise cultivation for producing a largenumber of microorganisms. A volume of the medium used in the agitatingcultivation without aeration is generally less than 1000 L, preferablyless than 500 L, more preferably less than 100 L. The lower limit of themedium is generally 0.8 L or more, preferably 4 L or more.

In the agitating cultivation without aeration, for example, a fermentorcapable of accomplishing sterility and capable of carrying out asmall-scale cultivation may be used. As such a fermentor, a small-sizedfermentor (such as a jar fermentor or a small-sized culture tank) havinga capacity of, for example, 1000 L or less, preferably 500 L or less,more preferably 100 L or less, most preferably less than 100 L, may beused. The lower limit of the capacity is generally 1 L or more,preferably 5 L or more.

When Matsutake V is produced by cultivating Matsutake IV, an agitatingcultivation is carried out without aeration in the liquid medium. Whencultivation on a small scale (for example, using a jar fermentor orsmall-sized culture tank having a capacity of less than 100 L) iscarried out with aeration, mycelia sometimes flocculate and lose growthpoints, and thus, sometimes lose viability as a seed culture.

In the initial stage of the agitating cultivation without aeration, theagitation is controlled by a agitation power per unit volume of aculture medium. Mycelia exhibit a rapid growth under agitation atgenerally 0.01 to 2 kW/m³, preferably 0.05 to 1 kW/m³. During the periodsubsequent to the initial stage, an oxygen supply is decreased by thegrowth of mycelia, and it becomes hard to disperse growing mycelia, andthus, it is necessary to appropriately increase a strength of theagitation.

Mycelia produced by the agitating cultivation without aeration may beseparated and collected in accordance with a conventional method, suchas filtration with a filter press, or centrifugation. The obtainedmycelia can be dried (or not dried), crushed, extracted, or formed inaccordance with the intended purpose as a final product.

(Submerged Cultivation)

The submerged cultivation may be started by inoculating a liquid mediumwith Matsutake V (i.e., Matsutake fungus obtained by the agitatingcultivation without aeration). Further, mycelia of a Matsutake fungus(for example, Matsutake V to Matsutake VII) obtained by the submergedcultivation can be used to repeat the submerged cultivation.Furthermore, Matsutake IV [i.e., Matsutake fungus obtained by theshaking cultivation (particularly, Matsutake fungus obtained by thestationary liquid cultivation, followed by the shaking cultivation)] canbe used in the submerged cultivation. Before the inoculation, theMatsutake mycelia used for inoculation can be homogenized.

In the submerged cultivation, air is forcefully supplied from theoutside of a fermentor to the culture medium. In addition, the airbubbles supplied to the medium are changed to microbubbles, bymechanical agitation with an agitator, or by a draft tube or a plate fordispersion, to enlarge a gas/liquid interface and prolong a residencetime of the bubbles in the culture medium. As a result, an efficientoxygen supply to microorganisms is accomplished in the submergedcultivation. The submerged cultivation may be carried out using, forexample, an aeration stirred fermentor, a bubble tower fermentor (anairlift fermentor), or a fluidized bed fermentor.

As the liquid medium used in the submerged cultivation, the same liquidmedium as that used in the agitating cultivation without aeration may beused. Further, the same substrates as used in the agitating cultivationwithout aeration may be used, and the concentrations of the substratesmay be adjusted in accordance with a desired yield. It is preferable toadjust an osmotic pressure derived from the substrates to 0.01 to 0.8MPa.

With respect to the temperature in cultivation, the propagation rate ininoculation, the concentration of initial mycelia, the period forcultivation, and the agitation power, the above explanations of theagitating cultivation without aeration are applicable to the submergedcultivation.

It is preferable that the submerged cultivation is carried out on alarge scale, i.e., by using a large-scale culture medium. A volume ofthe medium used in the submerged cultivation is not particularlylimited, so long as it does not exceed a range of a large-scalecultivation in an ordinary stepwise cultivation for producing a largenumber of microorganisms. A volume of the medium used in the submergedcultivation is generally 100 L or more, preferably 1000 L or more, morepreferably 3000 L or more.

In the submerged cultivation, for example, a fermentor capable ofaccomplishing sterility and capable of carrying out aeration (ifnecessary), submerged cultivation, and a large-scale cultivation may beused. As such a fermentor, a large-sized fermentor having a capacity of,for example, 100 L or more, preferably 1000 L or more, more preferably3000 L or more, may be used. In this connection, the term “large-sizedfermentor” as used herein includes a fermentor sometimes referred to asa “middle-sized fermentor”.

When the submerged cultivation on a large scale is carried out on anindustrial scale, for example, by using a large-sized fermentor havingan capacity of 100 L or more, aeration is carried out, if necessary, ata rate of preferably 0.05 to 1.0 vvm, more preferably 0.2 to 0.5 vvm.

In the initial stage of the submerged cultivation, the agitation iscontrolled by a agitation power per unit volume of a culture medium.Mycelia exhibit a rapid growth under agitation at generally 0.01 to 2kW/m³, preferably 0.05 to 1 kW/m³. During the period subsequent to theinitial stage, an oxygen supply is decreased by the growth of mycelia,and it becomes hard to disperse growing mycelia, and thus, it isnecessary to appropriately increase a strength of the agitation. In thesubmerged cultivation, preferably the cultivation at the initial stageis carried out with aeration at a low rate and under agitation at a lowrate, and the cultivation at the late stage is carried out with aerationat a high rate and under agitation at a high rate.

Mycelia produced by the submerged cultivation may be separated andcollected in accordance with a conventional method, such as filtrationwith a filter press, or centrifugation. The obtained mycelia can bedried (or not dried), crushed, extracted, or formed in accordance withthe intended purpose as a final product.

Hereinafter the present invention will be further explained by anembodiment using Tricholoma matsutake FERM BP-7304. The followingexplanations are generally applicable to Tricholoma matsutake strainsother than the FERM BP-7304 strain.

1. Properties in Growth

The FERM BP-7304 strain is sensitive to environmental factors. Physicalenvironmental factors include, for example, an osmotic pressure, or amechanical shock due to agitation. Because the growth requires a longtime, a large number of mycelia as a seed culture are needed in eachinoculation to finish the cultivation for short periods. Further, it isnecessary to establish a cultivation system capable of mass inoculation.

2. Basic Cultivation Conditions

(1) Resistance to Mechanical Shock

The main purpose of agitation in agitating cultivation with aeration isa dispersion of oxygen and nutrients. A growth rate of the FERM BP-7304strain is slow, and a demand for oxygen is small, and thus, anoversupply of oxygen is unnecessary. FIG. 1 is a graph showing therelationship between an agitation power and growth. When the agitationpower is strong, the growth of mycelia is inhibited. Because a decreasein dissolved oxygen is not observed, the main effect due to theagitation is considered a mechanical shock.

In addition, when the agitation power is strong, mycelia tend to becomepelleted, and thus, the growth is retarded. It is considered that theresults are caused by a flocculation effect by agitation. The mechanicaldamage to mycelia and the transformation into a pelleted form areimportant factors in the growth of the FERM BP-7304 strain.

(2) Growth and Osmotic Pressure

In the passway for utilizing carbon sources in basidiomycetes, it isgenerally considered that saccharides is decomposed into glucose, andfurther, glucose is converted into glucose-6-phosphate, and thenglucose-6-phosphate is incorporated into cells. That is, monosaccharidessuch as glucose are generally superior in efficiency to polysaccharides.In the FERM BP-7304 strain, it is considered that polysaccharides areutilized via glucose, but growth tends to be inhibited in a culturemedium containing a high concentration of glucose and exhibiting a highosmotic pressure. FIG. 2 shows the results of cultivation using culturemedia containing starch and glucose and exhibiting various osmoticpressures. When the concentration of glucose is high, the growth rate isslow, and the result suggests that the resistance to an osmotic pressureis low. When preparing a culture medium, a small amount of glucose isadded to a medium containing starch as a main carbon source, to fullyexploit the advantageous properties of two carbon sources.

(3) Required Amount of Seed Culture

It is known that Matsutake fungi grow slowly. Similarly, the FERMBP-7304 strain tends to grow slowly, but in an industrial production thegrowth should be finished by a predetermined period. FIG. 3 shows therelationship between a growth rate of the FERM BP-7304 strain and theamount of seed culture for inoculation. When the concentration ofinitial mycelia is approximately 0.1 to 2.0 g/L (including 0.05 to 2.0g/L of fibrous mycelia), the growth is rapid, and thus it is suggestedthat mass inoculation is necessary in an industrial cultivation.Particularly, when the amount of inoculation is small, the industrialcultivation tends to be difficult because a remarkably long inductionperiod is required.

(4) Control of Mycelia Form

In the cultivation of filamentous fungi it is known that the form ofmycelia significantly influences the growth. The FERM BP-7304 strain isbroadly classified into fibrous mycelia (FIG. 4) and pelleted mycelia(FIG. 5), and the fibrous mycelia are advantageous to the growth. FIG. 6shows growth curves in which pelleted and fibrous seed cultures are usedfor inoculation. When the pelleted seed culture was used forinoculation, the growth rate tends to be remarkably slow. Therefore, itis important in the industrial production to control the form of themycelia. As factors contributing to the form of the FERM BP-7304 strain,there may be mentioned, for example, the flow of a culture medium, theamount of seed culture for inoculation, or the composition of a culturemedium. Particularly, in a small-sized jar fermentor, mycelia tend to beentangled and fused with each other to become pelleted, due to the flowof a culture medium by aeration. Therefore, an optimization of aerationis preferable to obtain an excellent seed culture. Because pelletedmycelia do not grow efficiently as a seed culture, the content offibrous mycelia contained in a seed culture for industrial cultivationis preferably 50% ore more, most preferably 80%. In this connection, ifthe initial mycelia in the next stage contains approximately 0.05 to 2g/L (as dried weight) of fibrous mycelia, the content of fibrous myceliacontained in a seed culture is not limited to the above range.

3. Cultivation System

(1) Cultivation System for Seed Culture

As described above, the FERM BP-7304 strain tends to need a large amountof seed culture, and thus, it is preferable to use a system capable ofcultivating a large amount of seed culture. Therefore, the presentinventors established a method for cultivating a seed culture comprisingthe steps of cultivating a seed culture maintained on a solid mediumunder stationary conditions stepwisely, and carrying out shakingcultivation, as shown in FIG. 7. The purpose of the cultivation isacclimation to a liquid medium.

(2) Tank Cultivation System

As described above, the FERM BP-7304 strain tends to need a large amountof seed culture for a rapid growth of the FERM BP-7304 strain.Therefore, the concentration of initial mycelia in a tank cultivationsystem is preferably approximately 0.1 to 2.0 g/L. Further, mycelia inthe initial culture medium contain preferably approximately 0.05 to 2.0g/L (as the concentration of initial mycelia) of fibrous mycelia. FIG. 8shows a cultivation system capable of carrying out the process of thepresent invention. In the tank cultivation system, four stages ofcultivation including the main cultivation stage may be carrying out. Ineach stage, the propagation rate is set at approximately 5 to 15 timesto obtain approximately 0.1 to 2.0 g/L of the concentration of initialmycelia. According to the cultivation system and method shown in FIG. 8,approximately 12 to 14 g/L of mycelia can be produced by carrying outthe main cultivation for 12 to 14 days. FIG. 9 shows a growth curve inthe productive cultivation.

EXAMPLES

The present invention now will be further illustrated by, but is by nomeans limited to, the following Examples. In the Examples, a seedculture for a large-scale production was prepared, and the seed culturewas used to produce a large number of mycelia of Tricholoma matsutake.

Tricholoma matsutake FERM BP-7304 used in the Examples was deposited inthe International Patent Organism Depositary National Institute ofAdvanced Industrial Science and Technology [(Former Name) NationalInstitute of Bioscience and Human-Technology Agency of IndustrialScience and Technology (Address: AIST Tsukuba Central 6, 1-1, Higashi1-chome Tukuba-shi, Ibaraki-ken 305-8566 Japan)] on Sep. 14, 2000.Mycological features of Tricholoma matsutake FERM BP-7304 are describedin WO02/30440 (patent reference 1). For example, the strain ismaintained on 10 mL of an Ebios agar in a glass tube.

Hereinafter Tricholoma matsutake mycelia obtained by cultivation ofTricholoma matsutake FERM BP-7304 and corresponding to the aboveMatsutake II to VIII are referred to as Matsutake (II-1) to (VIII-1),respectively.

The following fermentors were used in each agitating cultivation step:

In Example 3, Comparative example 2-1, and Comparative example 3-1(without aeration), a 30-liter jar fermentor equipped with a sparger foraeration and an agitator (6 blade discs; 2 stages) was used in a statein which the sparger for aeration was removed therefrom, and referred toas “30-liter jar fermentor”.

In comparative example 3-1, the 30-liter jar fermentor equipped with asparger for aeration and an agitator (6 blade discs; 2 stages) was used,and referred to as “30-liter jar fermentor with a sparger”.

In Example 4, Examples 7-1 to 7-4, Examples 8-2 to 8-3, Examples 9-1 to9-3, and Example 11-1, a 200-liter fermentor equipped with a sparger foraeration and an agitator (4 blade discs; 2 stages) was used, andreferred to as “200-liter fermentor”.

In Example 5, a 7-m³ fermentor equipped with a sparger for aeration andan agitator (6 blade discs; 2 stages) was used, and referred to as “7-m³fermentor”.

In Example 6, a 65-m³ fermentor equipped with a sparger for aeration andan agitator (6 blade discs; 3 stages) was used, and referred to as“65-m³ fermentor”.

As to the expression “agitation power per unit volume of the culturemedium” in Examples 2 to 6, Example 7-1, Example 9-1, Examples 10-1 to10-3, and Example 11-1, the term “culture medium” in the expression doesnot mean only a prepared liquid medium, but also the whole contents(including a culture medium of the seed culture) contained in eachfermentor, such as 2-liter conical flask, 30-liter jar fermentor,30-liter jar fermentor with a sparger, 200-liter fermentor, 7-m³fermentor, or 65-m³ fermentor.

The following substrates for nutrient source used for preparing culturemedia by using the 200-L fermentor in Example 4, Example 7-1, Example9-1, or Example 11-1 are referred to as A-type substrates, B-typesubstrates, or C-type substrates, in necessary.

A-type substrates: (1) potato starch 4.9 kg, (2) glucose 140 g, (3)dried yeast extract 280 g, (4) potassium dihydrogen phosphate 280 g, (5)magnesium sulfate heptahydrate 42 g, (6) calcium chloride dihydrate 0.84g, (7) zinc sulfate heptahydrate 0.56 g, (8) manganese chloridetetrahydrate 0.70 g, (9) copper sulphate pentahydrate 0.14 g, (10) ironchloride hexahydrate 1.12 g, and (11) thiamin hydrochloride 0.14 g.

B-type substrates: The components and the contents thereof [(1), (2),and (4) to (11)] are the same as the A-type substrates, except for (3)dried yeast extract 320 g.

C-type substrates: The components (4) to (11) and the contents thereofare the same as the A-type substrates and B-type substrates. Further,dried yeast extract 640 g, and potato starch and glucose were contained.Amounts of potato starch and glucose are shown in Examples 11-1 to 11-3.

The procedures in which a production scale was enlarged from a 500-mLconical flask to a 65-m³ fermentor are illustrated in the followingExamples 1 to 6.

Example 1 Process for Producing Matsutake (III-1) by Stationary LiquidCultivation using 500-mL Conical Flask

Glucose (30 g/L) and dried yeast extract (3 g/L) were dissolved in tapwater to prepare a culture medium. An osmotic pressure of the mediumafter inoculation was 0.4 MPa. The medium was dispensed in 120 mLportions into 500-mL conical flasks, and sterilized. A broth (10 mL)containing Matsutake (II-1) maintained by stationary liquid cultivationwas used to inoculate the culture medium. Stationary liquid cultivationwas carried out at 23±2° C. for 180 days to produce a broth containingMatsutake (III-1) (5 g/L as dried weight). Propagation rate ininoculation: 13 times. Concentration of initial mycelia: 0.38 g/L.

Example 2 Process for Producing Matsutake (IV-1) by Shaking Cultivation

Glucose (30 g/L), dried yeast extract (1 g/L), soybean peptone (2 g/L),malt extract (1 g/L), potassium dihydrogen phosphate (1 g/L),dipotassium hydrogenphosphate (1 g/L), and magnesium sulfateheptahydrate (0.3 g/L) were dissolved in tap water to prepare a culturemedium. The medium was dispensed in 870 mL portions into 2-L conicalflasks, and sterilized. The broth (130 mL) containing Matsutake (III-1)(5 g/L as dried weight), obtained in Example 1, was homogenized and usedto inoculate the culture medium (870 mL). Shaking cultivation (anagitation power per unit volume of the culture medium by a rotaryshaker=0.14 kw/m³) was carried out at 23±2° C. for 20 days to produce abroth containing Matsutake (IV-1) (5 g/L as dried weight). In themycelia, 4.8 g/L of fibrous mycelia were contained. An osmotic pressureof the medium after inoculation was 0.4 MPa. Propagation rate ininoculation: 7.7 times. Concentration of initial mycelia: 0.65 g/L.

Example 3 Process for Producing Matsutake (V-1) in 30-L Jar Fermentor

In a 30-L jar fermentor, potato starch (600 g), glucose (60 g), driedyeast extract (40 g), potassium dihydrogen phosphate (40 g), magnesiumsulfate heptahydrate (6 g), calcium chloride dihydrate (0.12 g), zincsulfate heptahydrate (0.08 g), manganese chloride tetrahydrate (0.1 g),copper sulphate pentahydrate (0.02 g), iron chloride hexahydrate (0.18g), and thiamin hydrochloride (0.02 g) were dissolved in tap water, andsterilized to prepare a culture medium (18 L). An osmotic pressure ofthe medium after inoculation was 0.05 MPa.

The broth (2 L) containing Matsutake (IV-1) (5 g/L as dried weight),obtained in Example 2, was used to inoculate the culture medium.Cultivation (agitation power per unit volume of the culture medium=0.08kw/m³) was carried out without aeration to the medium from the spargerfor aeration at 23±2° C. for 7 days to produce a broth containingMatsutake (V-1) (1 g/L as dried weight). In the mycelia, 0.95 g/L offibrous mycelia were contained. Propagation rate in inoculation: 10times. Concentration of initial mycelia: 0.5 g/L (including 0.45 g/L offibrous mycelia).

Example 4 Process for Producing Matsutake (VI-1) in 200-L Fermentor

In a 200-L fermentor, the above A-type substrates were dissolved in tapwater, and sterilized to prepare a culture medium (120 L). An osmoticpressure of the medium was 0.05 MPa. The broth (20 L) containingMatsutake (V-1) (1 g/L as dried weight), obtained in Example 3, was usedto inoculate the culture medium. Submerged cultivation (an agitationpower per unit volume of the culture medium=0.12 kw/m³) was carried outwith aeration at a rate of 42 L/min at 23±2° C. for 7 days to produce abroth containing Matsutake (VI-1) (3 g/L as dried weight). In themycelia, 2.7 g/L of fibrous mycelia were contained. Propagation rate ininoculation: 7 times. Concentration of initial mycelia: 0.14 g/L(including 0.13 g/L of fibrous mycelia).

Example 5 Process for Producing Matsutake (VII-1) in 7-m³ Fermentor

In a 7-m³ fermentor, soluble starch (140 kg), glucose (4 kg), driedyeast extract (8 kg), potassium dihydrogen phosphate (8 kg), magnesiumsulfate heptahydrate (1.2 kg), calcium chloride dihydrate (24 g), zincsulfate heptahydrate (16 g), manganese chloride tetrahydrate (20 g),copper sulphate pentahydrate (4 g), iron chloride hexahydrate (32 g),and thiamin hydrochloride (4 g) were dissolved in tap water, andsterilized to prepare a culture medium (3.72 m³) An osmotic pressure ofthe medium was 0.05 MPa. The broth [280 L (corresponding to two tanks)]containing Matsutake (VI-1) (3 g/L as dried weight), obtained in Example4, was used to inoculate the culture medium. Submerged cultivation (anagitation power per unit volume of the culture medium=0.05 kw/m³) wascarried out with aeration at a rate of at 1.2 m³/min at 23±2° C. for 7days to produce a broth containing Matsutake (VII-1) (3 g/L as driedweight). In the mycelia, 2.6 g/L of fibrous mycelia were contained.Propagation rate in inoculation: 14.3 times. Concentration of initialmycelia: 0.21 g/L (including 0.19 g/L of fibrous mycelia).

Example 6 Process for Producing Matsutake (VIII-1) in 65-m³ Fermentor

In a 65-m³ fermentor, soluble starch (1575 kg), glucose (45 kg), driedyeast extract (135 kg), potassium dihydrogen phosphate (90 kg),magnesium sulfate heptahydrate (13.5 kg), calcium chloride dihydrate(270 g), zinc sulfate heptahydrate (180 g), manganese chloridetetrahydrate (225 g), copper sulphate pentahydrate (45 g), iron chloridehexahydrate (360 g), and thiamin hydrochloride (45 g) were dissolved intap water, and sterilized to prepare a culture medium (36 m³). Anosmotic pressure of the medium after inoculation was 0.05 MPa.

The broth [4 m³ (corresponding to one tank)] containing Matsutake(VII-1) (3 g/L as dried weight), obtained in Example 5, was used toinoculate the culture medium. Submerged cultivation was carried out withaeration at a rate of at 12 m³/min at 23±2° C. for 13 days to produce abroth containing Matsutake (VIII-1) (13.5 g/L as dried weight). Thecultivation was started at an agitation power per unit volume of theculture medium of 0.013 kw/m³, and the agitation power was increased, inaccordance with the growth of mycelia, to 0.12 kw/m³ on the 12th day ofthe cultivation. Propagation rate in inoculation: 10 times.Concentration of initial mycelia: 0.3 g/L (including 0.26 g/L of fibrousmycelia).

Comparative Example 1

Effects of the broth containing Matsutake (III-1) produced by stationaryliquid cultivation on shaking cultivation: Comparison with a broth notproduced via the stationary liquid cultivation step

The shaking cultivation for 20 days described in Example 2 was repeatedexcept that an Ebios agar (10 mL) containing Matsutake (II-1) was used.

However, the amount of dried mycelia contained in the broth obtainedafter the cultivation for 20 days could not be determined.

Example 7

Effects of a production period of the broth containing Matsutake (III-1)produced by stationary liquid cultivation on submerged cultivation inthe 200-L fermentor: Comparisons between the stationary liquidcultivation for 180 days in Example 1, and stationary liquid cultivationfor 30, 50, or 400 days

Example 7-1 Stationary Liquid Cultivation for 180 Days

The procedures described in Examples 1 to 3 were repeated to produce abroth containing Matsutake (V-1).

In a 200-L fermentor, the above B-type substrates were dissolved in tapwater, and sterilized to prepare a culture medium (120 L). The broth (20L) containing Matsutake (V-1) was used to inoculate the culture medium.Submerged cultivation (an agitation power per unit volume of the culturemedium=0.12 kw/m³) was carried out with aeration at a rate of 42 L/minat 23±2° C. for 12 days to produce a broth containing mycelia (12 g/L asdried weight). Propagation rate in inoculation: 7 times. Concentrationof initial mycelia: 0.14 g/L (including 0.13 g/L of fibrous mycelia).

Example 7-2 Stationary Liquid Cultivation for 30 days

The procedure described in Example 1 was repeated except that a periodof the stationary liquid cultivation was 30 days. Further, theprocedures described in Examples 2 and 3 were repeated to produce abroth. The cultivation in the 200-L fermentor described in Example 7-1was repeated except that the broth (20 L) was used to produce a brothcontaining mycelia (2 g/L as dried weight).

Example 7-3 Stationary Liquid Cultivation for 50 Days

The procedure described in Example 1 was repeated except that a periodof the stationary liquid cultivation was 50 days. Further, theprocedures described in Examples 2 and 3 were repeated to produce abroth. The cultivation in the 200-L fermentor described in Example 7-1was repeated except that the broth (20 L) was used to produce a brothcontaining mycelia (8 g/L as dried weight).

Example 7-4 Stationary Liquid Cultivation for 400 Days

The procedure described in Example 1 was repeated except that a periodof the stationary liquid cultivation was 400 days. Further, theprocedures described in Examples 2 and 3 were repeated to produce abroth. The cultivation in the 200-L fermentor described in Example 7-1was repeated except that the broth (20 L) was used to produce a brothcontaining mycelia (6 g/L as dried weight).

Comparative Example 2

Effects of the broth containing Matsutake (IV-1) produced by shakingcultivation on submerged cultivation: Comparison with a broth notproduced via the shaking cultivation step

Comparative Example 2-1 Not via the Shaking Cultivation Step

The broth (2 L) containing Matsutake (III-1) (5 g/L as dried weight),obtained in Example 1, was homogenized. The homogenized broth was usedto carry out cultivation in a 30-L jar fermentor in accordance with theprocedure described in Example 3. However, the amount of dried myceliacontained in the broth obtained after the cultivation for 7 days couldnot be determined.

In this connection, the results obtained via the shaking cultivation for20 days are shown in Example 7-1. The data in inoculation and aftercultivation were as follows: A broth containing mycelia (12 g/L as driedweight) was obtained. Propagation rate in inoculation: 7 times.Concentration of initial mycelia: 0.14 g/L (including 0.13 g/L offibrous mycelia).

Example 8

Effects of the broth containing Matsutake (IV-1) produced by shakingcultivation on submerged cultivation in the 200-L fermentor: Comparisonsbetween the shaking cultivation for 20 days in Example 2, and shakingcultivation for 5 or 3 days

Example 8-1 Shaking Cultivation for 20 Days

The results are shown in Example 7-1. The data in inoculation and aftercultivation were as follows: A broth containing mycelia (12 g/L as driedweight) was obtained. Propagation rate in inoculation: 7 times.Concentration of initial mycelia: 0.14 g/L (including 0.13 g/L offibrous mycelia).

Example 8-2 Shaking Cultivation for 5 Days

The procedure described in Example 2 was repeated using the brothproduced in Example 1, except that a period of the shaking cultivationwas 5 days. Further, the procedure described in Example 3 was repeatedto produce a broth. The broth was used to carry out cultivation in a200-L fermentor in accordance with the procedure described in Example7-1 to produce a broth containing mycelia (7 g/L as dried weight).

Example 8-3 Shaking Cultivation for 3 Days

The procedure described in Example 2 was repeated using the brothproduced in Example 1, except that a period of the shaking cultivationwas 3 days. Further, the procedure described in Example 3 was repeatedto produce a broth. The broth was used to carry out cultivation in a200-L fermentor in accordance with the procedure described in Example7-1 to produce a broth containing mycelia (0.5 g/L as dried weight).

Comparison Example 3

Effects of the broth containing Matsutake (V-1) produced by agitatingcultivation without aeration in the 30-L jar fermentor, on submergedcultivation in the 200-L fermentor: Comparison with a broth producedwith aeration in a 30-liter jar fermentor with a sparger

Comparison Example 3-1 Cultivation of Matsutake (IV-1) with Aeration

The procedure described in Example 3 was repeated using the brothproduced in accordance with the procedures described in Examples 1 and2, except that the 30-liter jar fermentor with a sparger was used andthat aeration at a rate of 2 L/min was carried out, to produce a broth.Mycelia in the broth flocculated. The amount of dried mycelia after thecultivation was 0.6 g/L (including 0.03 g/L of fibrous mycelia).Further, the broth was used to carry out cultivation in a 200-Lfermentor in accordance with the procedure described in Example 7-1 toproduce a broth containing mycelia (5 g/L as dried weight).

In this connection, the results obtained without aeration in thecultivation of Matsutake (IV-1) are shown in Example 7-1. As withExample 3, most mycelia in the broth produced in the 30-liter jarfermentor without aeration were fibrous. The amount of dried myceliaafter the cultivation was 1 g/L (including 0.95 g/L of fibrous mycelia).The data in inoculation and after cultivation were as follows: A brothcontaining Mycelia (12 g/L as dried weight) was obtained. Propagationrate in inoculation: 7 times. Concentration of initial mycelia: 0.14 g/L(including 0.13 g/L of fibrous mycelia).

Example 9

Effects of a volume of the broth containing Matsutake (VI-1) produced bysubmerged cultivation with aeration on submerged cultivation in the200-L fermentor: Comparisons between various volumes of broths used inExample 4

Example 9-1 Inoculation with 10 L of Broth Containing Matsutake (VI-1)

The procedures described in Examples 1 to 4 were repeated to produce abroth containing Matsutake (VI-1).

In a 200-L fermentor, the B-type substrates were dissolved in tap water,and sterilized to prepare a culture medium (130 L). The broth (10 L)containing Matsutake (VI-1) was used to inoculate the culture medium(130 L). Submerged cultivation (an agitation power per unit volume ofthe culture medium=0.12 kw/m³) was carried out with aeration at a rateof 42 L/min at 23±2° C. for 12 days to produce a broth containingmycelia (12 g/L as dried weight). Propagation rate in inoculation: 14times. Concentration of initial mycelia: 0.21 g/L (including 0.19 g/L offibrous mycelia).

Example 9-2 Inoculation with 5 L of Broth Containing Matsutake (VI-1)

The cultivation in the 200-L fermentor was carried out in accordancewith the procedure described in Example 9-1, except that the broth (5 L)containing Matsutake (VI-1) produced by the procedures described inExamples 1 to 4 was used together with sterile water (5 L), to produce abroth containing mycelia (10 g/L as dried weight). Propagation rate ininoculation: 28 times. Concentration of initial mycelia: 0.107 g/L(including 0.097 g/L of fibrous mycelia).

Example 9-3 Inoculation with 2 L of Broth Containing Matsutake (VI-1)

The cultivation in the 200-L fermentor was carried out in accordancewith the procedure described in Example 9-1, except that the broth (2 L)containing Matsutake (VI-1) produced by the procedures described inExamples 1 to 4 was used together with sterile water (8 L), to produce abroth containing mycelia (2 g/L as dried weight). Propagation rate ininoculation: 70 times. Concentration of initial mycelia: 0.043 g/L(including 0.04 g/L of fibrous mycelia).

Example 10

Effects of agitating conditions in submerged cultivation by using, as aseed culture, the broth containing Matsutake (VI-1) produced bysubmerged cultivation with aeration, with respect to mycelia growth inthe 200-L fermentor:

Example 10-1 Agitation Power per Unit Volume of a Culture Medium of 0.12kW/m³

The procedures described in Example 9-1 were repeated. The data ininoculation and after cultivation were as follows: A broth containingmycelia (12 g/L as dried weight) was obtained. Propagation rate ininoculation: 14 times. Concentration of initial mycelia: 0.21 g/L(including 0.19 g/L of fibrous mycelia).

Example 10-2 Agitation Power per Unit Volume of a Culture Medium of 1.09kW/m³

The procedures described in Example 10-1 were repeated, except that theagitation power per unit volume of a culture medium was 1.09 kW/m³ whencarrying out submerged cultivation for 12 days. A broth containingmycelia (7 g/L as dried weight) was obtained. Propagation rate ininoculation: 14 times. Concentration of initial mycelia: 0.21 g/L(including 0.19 g/L of fibrous mycelia).

Example 10-3 Agitation Power per Unit Volume of a Culture Medium of 2.63kW/m³

The procedures described in Example 10-1 were repeated, except that theagitation power per unit volume of a culture medium was 2.63 kW/m³ whencarrying out submerged cultivation for 12 days. A broth containingmycelia (7 g/L as dried weight) was obtained. However, the amount ofdried mycelia contained in the broth obtained after the cultivation for12 days could not be determined.

Example 11

Effects of various carbon sources in submerged cultivation by using, asa seed culture, the broth containing Matsutake (VI-1) produced bysubmerged cultivation with aeration, with respect to mycelia growth inthe 200-L fermentor: Comparison between the amount of potato starch andthat of glucose (as an osmotic pressure)

Example 11-1 Osmotic Pressure of 0.05 MPa

The procedures described in Examples 1 to 4 were repeated to produce abroth containing Matsutake (VI-1).

In a 200-L fermentor, the C-type substrates, 9.8 kg of potato starch,and 140 g of glucose were dissolved in tap water, and sterilized toprepare a culture medium (130 L) having an osmotic pressure of 0.05 MPa.The broth (10 L) containing Matsutake (VI-1) was used to inoculate theculture medium (130 L). Submerged cultivation (an agitation power perunit volume of the culture medium=0.12 kw/m³) was carried out withaeration at a rate of 42 L/min at 23±2° C. for 12 days to produce abroth containing mycelia (14 g/L as dried weight). Propagation rate ininoculation: 14 times. Concentration of initial mycelia: 0.21 g/L(including 0.19 g/L of fibrous mycelia).

Example 11-2 Osmotic Pressure of 0.50 MPa

The procedures described in Example 11-1 were repeated, except that 4.97kg of potato starch and 4.97 kg of glucose were used to prepare aculture medium (130 L) having an osmotic pressure of 0.50 MPa, whencarrying out submerged cultivation for 12 days. A broth containingmycelia (9.5 g/L as dried weight) was obtained. Propagation rate ininoculation: 14 times. Concentration of initial mycelia: 0.21 g/L(including 0.19 g/L of fibrous mycelia).

Example 11-3 Osmotic Pressure of 0.98 MPa

The procedures described in Example 11-1 were repeated, except that 140g of potato starch and 9.8 kg of glucose were used to prepare a culturemedium (130 L) having an osmotic pressure of 0.98 MPa, when carrying outsubmerged cultivation for 12 days. A broth containing mycelia (2 g/L asdried weight) was obtained. Propagation rate in inoculation: 14 times.Concentration of initial mycelia: 0.21 g/L (including 0.19 g/L offibrous mycelia).

Example 12

Effects of agitation power in agitating cultivation on form and growthactivity of Matsutake (V):

Example 12-1 Agitation Power of 1.6 kw/m³

The procedures described in Example 2 were repeated, except that theagitation power per unit volume of a culture medium in the shakingcultivation was 1.6 kw/m³, to produce a broth containing Matsutake(IV-1) (3.2 g/L as dried weight) including 2.2 g/L of fibrous mycelia.Then the procedures described in Example 3 were repeated, except that 3L (corresponding to the concentration of initial mycelia in Example 3)of the broth containing Matsutake (IV-1) were used for inoculation, toproduce a broth containing Matsutake (V-1) in the 30-L jar fermentor. Inthe broth, mycelia (0.75 g/L as dried weight) including 0.6 g/L offibrous mycelia were contained. Propagation rate in inoculation: 6.7times. Concentration of initial mycelia: 0.48 g/L (including 0.38 g/L offibrous mycelia).

The Matsutake (V) was used to repeat the procedures described in Example7-1, except that 20 L of the Matsutake (V) was used for inoculation inthe 200-L fermentor. As a result, a broth containing mycelia (9.7 g/L asdried weight) was obtained.

Example 12-2 Agitation Power of 2.2 kw/m³

The procedures described in Example 2 were repeated, except that theagitation power per unit volume of a culture medium in the shakingcultivation was 2.2 kw/m³ (i.e., a high speed agitation), to produce abroth containing Matsutake (IV-1) (2.4 g/L as dried weight) including0.013 g/L of fibrous mycelia. Then the procedures described in Example 3were repeated, except that 4 L (corresponding to the concentration ofinitial mycelia in Example 3) of the broth containing Matsutake (IV-1)were used for inoculation, to produce a broth containing Matsutake (V-1)in the 30-L jar fermentor. In the broth, mycelia (0.5 g/L as driedweight) including 0.01 g/L of fibrous mycelia were contained.Propagation rate in inoculation: 5 times. Concentration of initialmycelia: 0.48 g/L (including 0.0026 g/L of fibrous mycelia).

The Matsutake (V) was used to repeat the procedures described in Example7-1, except that 20 L of the Matsutake (V) was used for inoculation inthe 200-L fermentor. As a result, a broth containing mycelia (4.7 g/L asdried weight) was obtained.

Example 13

Effects of agitation power for Matsutake (VI) in 200-L fermentor on formand growth activity of Matsutake (VI):

Example 13-1 Agitation Power of 1.7 kw/m³

The procedures described in Examples 1 to 3 were repeated to produce abroth containing Matsutake (V-1). The procedures described in Example 4were repeated, except that the agitation power per unit volume of aculture medium in the shaking cultivation was 1.7 kw/m³ in the 200-Lfermentor, to produce a broth containing Matsutake (VI-1) (1.6 g/L asdried weight) including 1.1 g/L of fibrous mycelia. The Matsutake (VI-1)was used to repeat the procedures described in Example 9-1 to obtain abroth containing mycelia (9.7 g/L as dried weight). Propagation rate ininoculation: 14 times. Concentration of initial mycelia: 0.11 g/L(including 0.78 g/L of fibrous mycelia).

Example 13-2 Agitation Power of 2.2 kw/m³

The procedures described in Examples 1 to 3 were repeated to produce abroth containing Matsutake (V-1). The procedures described in Example 4were repeated, except that the agitation power per unit volume of aculture medium in the shaking cultivation was 2.2 kw/m³ in the 200-Lfermentor, to produce a broth containing Matsutake (VI-1) (0.68 g/L asdried weight) including 0.02 g/L of fibrous mycelia. The proceduresdescribed in Example 9-1 were repeated, except that 25 L of the obtainedMatsutake (VI-1) was used for inoculation, and that 115 L of medium wasused to obtain a broth containing mycelia (2.5 g/L as dried weight).Propagation rate in inoculation: 5.6 times. Concentration of initialmycelia: 0.12 g/L (including 0.0036 g/L of fibrous mycelia).

Example 14 Evaluation of Tricholoma matsutake mycelia Obtained in MassCultivation for Natural Killer (NK) Cell Activity

(1) Preparation of Lyophilized Tricholoma matsutake mycelia Obtained byMass Cultivation

The Tricholoma matsutake mycelia obtained in Example 6 was centrifugedby a basket centrifuge), and the collected cake was crushed into smallblocks having a length of approximately 5 mm. A tray (510 mm×790 mm) forlyophilization was filled with 6 kg of the crushed blocks of Tricholomamatsutake mycelia [water content=79.4% wet base (W.B.)], and prefrozenat −35° C. for 24 hours. The prefrozen mycelia were lyophilized under areduced pressure of 13 Pa to obtain 1.2 kg of lyophilized mycelia. Inthe lyophilization, the maximum temperature was 50° C. and the timerequired for the lyophilization was 24 hours. The water content of thelyophilized mycelia was 2.7% W.B.

The resulting lyophilized mycelia (1.2 kg) was crushed by a crusher (pinmill; 5000 r/min) to obtain powder. In the crushing treatment, thelyophilized mycelia were supplied at a rate of 12 kg/h, and thetemperature was controlled at 50° C. or less. The particle size of thepowder was such that 90% thereof passed through a mesh filter having amesh opening of 125 μm.

(2) Evaluation for NK Cell Activity

In this Example, an NK cell activity was evaluated as a physiologicalactivity of Tricholoma matsutake mycelia. The NK cell activity is knownas an index of an activity of promoting a recovery from stress. The NKcell activity was measured in accordance with a method (an evaluationsystem using mice) described in Evaluation Example 1 of WO02/30440. Moreparticularly, “Lytic Units 30% (LU30)”, that is, the number of cellswhich kill 30% tumor cells per 10⁷ cells of effector cells, wascalculated, and the obtained values were compared by t-test. In theexperiment, a dose of Tricholoma matsutake mycelia power was 150mg/kg/day per mouse (corresponding to 0.6 g/day in human).

As a result, lyophilized powder of Tricholoma matsutake mycelia preparedin Example 14 (1) exhibited the NK cell activity, and a significantdifference (P<0.05) with respect to a control group in which distilledwater was administered was confirmed.

INDUSTRIAL APPLICABILITY

According to the process of the present invention, a large number ofMatsutake mycelia can be produced without a loss of physiologicalactivities. For example, Matsutake mycelia as a seed culture can beproduced by cultivating mycelia cultivated or maintained in a solid orliquid medium, in a liquid medium under stationary conditions, andcultivating the obtained mycelia under shaking, and further cultivatingthe mycelia on a small scale (for example, by using a small-sizedfermentor having a capacity of less than 100 L) under agitation withoutaeration or with aeration at a low rate of less than 0.05 vvm in aliquid medium. Further, the obtained seed culture can be cultivatedunder submerged conditions on a large scale (for example, by using amiddle-sized or large-sized fermentor) to produce a large number ofMatsutake mycelia.

Although the present invention has been described with reference tospecific embodiments, various changes and modifications obvious to thoseskilled in the art are possible without departing from the scope of theappended claims.

1. A process for producing a mycelium of a Matsutake fungus, comprisingthe step of cultivating a mycelium on a small scale under agitationwithout aeration in a liquid medium or with aeration at a low rate ofless than 0.05 vvm.
 2. The process according to claim 1, furthercomprising, as a precultivation step before the agitating cultivationstep, (1) cultivating a mycelium in a liquid medium under stationaryconditions, (2) cultivating a mycelium under shaking, or (3) cultivatinga mycelium in a liquid medium under stationary conditions, and furthercultivating the obtained mycelium under shaking.
 3. The processaccording to claim 1, wherein an agitation power per unit volume of aculture medium in the agitating cultivation step is 0.01 to 2 kW/m³. 4.A process for producing a mycelium of a Matsutake fungus, comprising thesteps of: cultivating a mycelium in a liquid medium under stationaryconditions, and cultivating the obtained mycelium under shaking.
 5. Aprocess for producing a mycelium of a Matsutake fungus, comprising thestep of cultivating the mycelium obtained by the process according toclaim 1 as a seed culture under submerged conditions.
 6. The processaccording to claim 2, wherein a period of the stationary cultivationstep is 30 to 400 days.
 7. The process according to claim 2, wherein aperiod of the shaking cultivation step is 5 to 50 days.
 8. The processaccording to claim 1, wherein a propagation rate in inoculation is 2 to50 times.
 9. The process according to any claim 1, wherein an osmoticpressure of a culture medium is 0.01 to 0.8 MPa.
 10. The processaccording to claim 1, wherein a concentration of a fibrous myceliumcontained in an initial mycelium is 0.05 g/L or more.
 11. The processaccording to claim 2, wherein an agitation power per unit volume of aculture medium in the agitating cultivation step is 0.01 to 2 kW/m³. 12.A process for producing a mycelium of a Matsutake fungus, comprising thestep of cultivating the mycelium obtained by the process according toclaim 2 as a seed culture under submerged conditions.
 13. A process forproducing a mycelium of a Matsutake fungus, comprising the step ofcultivating the mycelium obtained by the process according to claim 3 asa seed culture under submerged conditions.
 14. A process for producing amycelium of a Matsutake fungus, comprising the step of cultivating themycelium obtained by the process according to claim 4 as a seed cultureunder submerged conditions.
 15. The process according to claim 3,wherein a period of the stationary cultivation step is 30 to 400 days.16. The process according to claim 3, wherein a period of the stationarycultivation step is 30 to 400 days.
 17. The process according to claim4, wherein a period of the stationary cultivation step is 30 to 400days.
 18. The process according to claim 3, wherein a period of theshaking cultivation step is 5 to 50 days.
 19. The process according toclaim 4, wherein a period of the shaking cultivation step is 5 to 50days.
 20. The process according to claim 5, wherein a period of theshaking cultivation step is 5 to 50 days.